1. Field of the Invention
The present invention relates to an oxygen carrier comprising hemoglobin vesicles, and more particularly, to an oxygen carrier system having a reducing agent capable of reducing methemoglobin to which hemoglobin has been oxidized.
2. Description of the Related Art
In a current blood transfusion system, there are pointed out problems that 1) there is a possibility of infection (hepatitis, AIDS virus etc.), 2) a storing time limit for erythrocyte is about 3 weeks, 3) with advent of an aging society, a ratio of aged person among blood transfused patients is increased, while a total of healthy blood donors continues to be decreased, 4) there is a risk of contamination during storage of erythrocyte, 5) blood transfusion can not be applied to a patient who rejects blood transfusion for the religious reasons, and 6) the system can not be applied to emergent demand at disaster.
Therefore, demand for blood cell substitutes which can instantly respond at any time anywhere regardless of a blood group is increasing. As one of substitutes, transfusion preparations such as an electrolyte transfusion and a colloidal transfusion have been widely used, but these transfusion preparations do not have a function of erythrocyte of carrying oxygen which is the most important function of blood. Therefore, exploitation of a transfusion having oxygen carrying ability (oxygen infusion) is urgently demanded.
Recently, exploitation of an oxygen infusion using hemoglobin obtained by purifying hemoglobin (e.g., human hemoglobin, bovine hemoglobin, recombinant hemoglobin) which binds to and dissociates oxygen in erythrocyte, and chemically modifying the purified hemoglobin, is being progressed. Intermolecular crosslinked (polymerized) hemoglobin, water-soluble polymer-bound hemoglobin, and intermolecular crosslinked polymerized hemoglobin have been subjected to a clinical trial in Europe and USA. However, regarding an oxygen infusion using these chemically modified hemoglobins, various side effects due to naked hemoglobin having no cell-type structure as erythrocyte have been pointed out. Accompanied with this, importance of a cell-type structure in which hemoglobin is encapsulated into a vesicle has been revealed.
Since it was found that phospholipid which is a biomembrane component alone forms a vesicle, and in Djordjevich et al., Fed. Proc. 36, 567, 1977, a system in which hemoglobin is encapsulated in a vesicle composed of phospholipid/cholesterol/fatty acid (hemoglobin vesicle) was studied, some groups including a group of the present inventors intensively studied a hemoglobin vesicle. A hemoglobin vesicle has advantages that 1) hemoglobin is not modified, 2) a viscosity, a colloidal osmotic pressure and an oxygen affinity can be adjusted to an arbitrary value, 3) a retention time in blood circulation is extended, and 4) various additives can be encapsulated into an aqueous phase in the vesicle at an arbitrary concentration. A group of the present inventors has previously established independently an effective process for preparing a hemoglobin vesicle, obtained a hemoglobin vesicle dispersion having various physical property values extremely near those of blood, and confirmed excellent oxygen carrying ability and safety thereof also in an animal experiment (Tsuchida ed., Blood Substitutes: Present and Future Perspective, Elsevier, Amsterdam, 1998).
Hemoglobin contains four hemes, and when the central iron is divalent (Fe2+), hemoglobin can reversibly bind to oxygen, and becomes oxyhemoglobin under atmospheric air. On the other hand, methemoglobin in which a central iron becomes electron oxidation-type trivalent (Fe3+) can not bind to oxygen. In addition, superoxide anion and hydrogen peroxide are produced accompanied with conversion of oxyhemoglobin to methemoglobin, and they act as an oxidizing agent to promote production of methemoglobin. There are methemoglobin reductase and active oxygen extinguishing enzyme in erythrocyte, and mechanism of maintaining a methemoglobin content at a level of 1% or lower is exerted. However, in an oxygen infusion using hemoglobin, since these enzymes are removed at purification of hemoglobin, hemoglobin is easily oxidized during storage and after administration, and oxidized hemoglobin (methemoglobin) is not reduced, thereby extinguishing oxygen carrying ability.
In order to reduce this methemoglobin, various methods have been proposed. For example, a method of encapsulating a reducing agent such as glutathione, homocysteine and ascorbic acid, or active oxygen extinguishing enzyme such as catalase and superoxide dismutase in a hemoglobin vesicle (Sakai et al., Bull. Chem. Soc. Jpn., 1994 and Takeoka et al., Bioconjugate Chem., 8, 539–544, 1997), a method of introducing methylene blue as an electron transporting substance into a vesicle membrane, and reducing hemoglobin in a vesicle by an electron transport mechanism from NADH added to an external aqueous phase of a vesicle (Takeoka et. al., Bull. Chem. Soc. Jpn., 70, 1171–1178, 1997), and a method of reducing methemoglobin by irradiation with the light in a near ultraviolet region (Sakai et al., Biochemistry, 39, 14595–14602, 2000) have been tried.
Among the aforementioned reducing methods, in a method of encapsulating a reducing agent such as glutathione and homocysteine together with purified hemoglobin into a vesicle, since a reducing agent is auto-oxidized and inactivated more rapidly than a rate of auto-oxidizing hemoglobin into methemoglobin, efficacy is low. In addition, a reducing agent produces active oxygen such as superoxide anion and hydrogen peroxide at the auto-oxidation, and this converts oxyhemoglobin to methemoglobin or ferrylhemoglobin.
Accordingly, an object of the present invention is to provide a system which uses a reducing agent, but further effectively reduces methemoglobin in a hemoglobin vesicle.